Post-transcriptional regulation of messenger RNA (mRNA) stability and translation are important control points for gene expression. The overall goal of this project is to generate and utilize structural information to enhance our understanding of these processes. One mechanism for regulation of mRNA stability involves the presence of adenosine-uridine (AU)-rich elements (AREs) in the 3? untranslated regions of many short-lived mRNAs. These AREs are found in mRNAs encoding proto-oncogenes, cytokines, and lymphokines and mediate their rapid degradation. The Hu proteins, human homologues of the Drosophila protein ELAV (embryonic lethal, abnormal visual), bind to AREs and stabilize the mRNAs to which they bind. One of the objectives of this project is to determine the three-dimensional structures of Hu protein RNA recognition motif (RRM) domains bound to RNA elements to understand how Hu proteins specifically recognize their RNA partners, examine how the protein-RNA interactions may stabilize the mRNA, and provide insight into how over 1000 proteins utilize the RRM protein scaffold to recognize different RNA sequences. We determined a 1.8 ? crystal structure of an ARE-binding fragment of HuD in complex with a fragment of the cfos ARE, a class I ARE, and a 2.3 ? crystal structure of the same fragment of HuD bound to a minimal fragment of the tumor necrosis factor a ARE, a class II ARE. These structures revealed a consensus RNA recognition sequence for HuD that highlights the importance of some bases, but the relative lack of specificity at other positions. Comparison to structures of other proteins containing RRM domains revealed that although these domains bind to different RNA structures (e.g. single-stranded RNA, stem-loop RNA) and sequences, a common binding mode using two nucleotide binding pockets is observed in all RRM domain structures. We also determined the structure of a fragment of HuD protein that was slightly longer at the C-terminus, but it did not reveal any additional contact with the RNA. We also have begun studying the post-transcriptional regulation by Puf family proteins. This family of proteins are found in organisms from humans to yeast and contain a conserved RNA-binding domain, the PUMILIO-homology domain (PUM-HD). Several family members have been shown to regulate the stability or repress the translation of their target mRNAs. We have determined the crystal structure of the PUM-HD from the human PUMILIO protein. Approximately 80% of the amino acid positions in the PUM-HD are identical to those in the Drosophila PUMILIO PUM-HD. The Drosophila PUMILIO protein is involved in early embryonic patterning and has been shown to repress the translation of maternal hunchback mRNA in concert with NANOS and BRAIN TUMOR proteins by specifically binding to Nanos Response Element (NRE) sequences in the 3? untranslated region. The function of the human protein is not known. The crystal structure of the PUM-HD resembles a half-doughnut. The distribution of sidechains on the inner and outer faces of this half-doughnut suggests that the inner face binds RNA while the outer surface interacts with proteins such as NANOS and BRAIN TUMOR.